1. Field of the Invention
The present invention relates to the fields of cytopathology and histology and, more specifically, to an improved cytologic and histologic fixative formulation for fixing cells, cell aggregates and small tissue fragments for the examination of same and for the diagnosis of disease.
2. Description of the Prior Art
Properly fixing (i.e., preserving) cytologic material such as cells, cell aggregates and small tissue fragments derived from cytologic collections of human or animal tissue is a prerequisite to the accurate diagnosis of disease, especially cancer. Cytologic material must be fixed as soon as possible after obtaining the material to prevent cell distortion.
Cytologic specimens, which constitute the examinable form of the cytologic material, may be prepared by well-understood smear or fluid techniques. Because there may be a considerable lapse of time before these specimens are further processed by staining, coverslipping, and so forth, however, it is important to apply a fixative to the cytologic material as a means of preserving and fixing the cells.
Air-dried and tetrachrome-dye stained cytologic specimens, although popular abroad, are not generally used in the United States. Rather, wet fixation, either by the immersion of slides into an alcohol solution, by saturation of slides with a spray fixative or by directly discharging cytologic material into an alcohol solution, is a knowm method of cell fixation. Cell fixation is a prerequisite for interpretable Papanicolaou, Hematoxylin and Eosin or other stained cytologic specimen slides.
Generally, alcohol solutions, with or without other additives such as polyethylene glycol, ranging from 50% to 95% (v/v: methanol, ethanol, isopropanol) are known solutions for use in wet fixation. When alcohol solutions greater than 50% (v/v) are used for collecting and fixing fluids high in protein, however, a protein sediment forms which subsequently hardens. Protein sedimentation makes the fixed cytologic material difficult to transfer to glass slides for examination, regardless of whether the transfer is done by direct application to the glass slide, by cytofiltration through a small pore filter, or by cytocentrifugation onto glass slides coated with an adhesive such as chrome alumn gelatin.
The preparation of a technically satisfactory cytologic specimen usually requires the skill of a trained cytologist, cytotechnologist or physician. See generally, Zajicek, J., "The Aspiration Biopsy Smear" in Diagnostic Cytology and its Histopathologic Bases, 3rd Ed. Koss, L. G., Editor (Philadelphia: J. B. Lippincott), Vol. 2, Chapter 29. According to the Zajicek article, when the collection of cytologic material is performed by suspending the cytologic material in an alcohol solution, however, less skill is required and technical problems are diminished.
Cytologic material with a high red blood cell content dilutes the cell population of diagnostic interest by red blood cells. Methods have been used to decolorize the red blood cells in such cytologic specimens such as post-fixation of the cytologic specimen slide in Carnoy's solution comprising 60% ethanol, 30% chloroform and 10% glacial acetic acid (v/v). Such post-fixation of the cytologic specimen slide creates the additional problem of diluting the number of diagnostic cells on the cytologic specimen slide.
Many cytologic specimens such as fine needle aspiration biopsies, brushings or scrapings of tissues and other such specimens consist of admixed tissue fragments and cells. Alcohol fixatives are not optimum fixatives for cytologic specimens containing tissue fragments because the processed tissue fragments become distorted in their appearance. Indeed, there is an advantage to examining both cells and tissue fragments obtained from cytologic specimens. See, Maksem, J. A., et al. "Aspiration Cytology and Transmission Electron Microscopy (TEM): A Novel Tissue Collection Technique With Useful Diagnostic Applications," Proceedings E.M.S.A., 42, 90-93 (1984). See also, Yamamoto, R., et al. "Histocytologic Diagnosis of Pancreatic Cancer by Percutaneous Aspiration Biopsy Under Ultrasonic Guidance," Am. J. Clin. Pathol., 83, 409-414 (1985).
Other sources have reported the advantages of working with cells collected in suspension. See Alfthan, O., et al., "Cytological Aspiration Biopsy and Vim-Silverman Biopsy in the Diagnosis of Prostatic Carcinoma," Chir. Gynaec. Fenn., 59 226-229 (year). See also, Smith, M. J., et al., "Fine Needle Aspiration and Endoscopic Brush Cytology," Acta Cytol., 456-459 (1980); Saccomanno, G., Diagnostic Pulmonary Cytology (Chicago: American Society of Clinical Pathologists) (1978).
Moreover, alcohol-free suspensions which are the most versatile cannot be stored for a long amount of time or transported over long distances. See, Boon, M. E. and Lykles, C., "Imaginative Approach to Fine Needle Aspiration Cytology," Lancet, 1031-1032 (1980).
It is thus an object of this invention to provide a cytologic and histologic fixative formulation that fixes and preserves cells, cell aggregates and small tissue fragments in a liquid suspension.
It is yet another object of this invention to provide a fixative formulation that minimizes protein precipitation in the liquid suspension.
It is indeed another object of this invention to provide a fixative formulation that selectively eliminates or reduces red blood cell contamination of cytologic material.
It is still another object of this invention to provide a fixative formulation that retains tissue samples which are incidentally collected along with cytologic material for further histological processing.
It is yet a further object of this invention to provide a fixative formulation that allows shipment of the liquid suspension of cells, cell aggregates and tissue fragments under conditions typically encountered in postal carriage, permitting remote users without available cytologists, cytotechnologists, physicians or other personnel experienced in the preparation of cytologic samples to fix a cytologic specimen for later processing, and whereby technically satisfactory cytologic sample slides may be produced therewith.